非蛋白质氨基酸的酶促合成及其作为中间体参与新药合成的研究
发布时间：2019-05-14 15:05
【摘要】：O-乙酰丝氨酸（硫醇）裂解酶(O-acetylserine sulfhydrylase, OASS)是大肠杆菌硫酸盐同化途径及半胱氨酸合成中重要的生物酶之一，和丝氨酸乙酰转移酶(Serineacetyltransferase, SAT)以酶复合体的形式存在，通常被称为半胱氨酸合酶(Cysteinesynthase)。在微生物和植物细胞内，OASS和SAT将环境中的无机硫还原成-2价的硫，经二步酶催化途径由L-丝氨酸合成L-半胱氨酸。首先以丝氨酸和乙酰辅酶A为底物，在SAT催化下，合成O-乙酰丝氨酸(O-acetylserine, OAS)和辅酶A；然后在OASS的催化下，通过硫化物将OAS转化为半胱氨酸。通过替代第二步反应中底物，OASS能够利用各种亲核试剂合成出非蛋白质氨基酸。 非蛋白质氨基酸是自然界存在的蛋白质中发现的20种氨基酸以外的其他氨基酸，因其丰富了肽链的多样性，为跨越20种天然氨基酸屏障，人工合成蛋白质提供了基础材料，是国内外合成肽类、类肽类药物的主要成分，也是合成手性药物的极具吸引力的侯选基础材料，是众多新药的关键中间体，在科研和医药领域用途广泛。 本论文以大肠杆菌Escherichia coli (E. coli) DH5α基因组DNA为模板，PCR扩增得到大小为972bp的O-乙酰丝氨酸（硫醇）裂解酶A (OASS-A)基因cysK ORF区基因片段，将目的片段与表达载体pET-22b(+)相连，得到重组质粒CysK-pET-22b(+)，转化到表达菌中，筛选诱导表达条件，进行蛋白纯化，获得高浓度可溶的OASS-A重组蛋白。以O-乙酰丝氨酸(OAS)为底物，对得到的重组蛋白进行酶活性检测，测得纯化的重组蛋白的活性单位为750U/mg，酶的粗提液中重组蛋白的活性单位为400U/mg。以重组OASS-A作为酶催化剂，O-乙酰丝氨酸和苯硫酚作为底物，合成非蛋白质氨基酸S-苯基-L-半胱氨酸(S-phenyl-L-cysteine, S-P-C)，通过高效液相色谱和核磁共振技术对产物结构进行鉴定，得到高纯度的S-P-C。建立了通过得到大量的活性稳定的重组酶作为生物催化剂，高效率合成非蛋白质氨基酸的有效模式。根据药物拼接原理，，将S-P-C与活性化合物积雪草酸、脱氧胆酸的有效基团进行偶联，对产物进行结构鉴定，并初步检测合成产物对癌细胞增殖影响初步检测，MTT实验结果显示，合成的终产物均具有一定的增殖抑制作用。
[Abstract]:O-acetylserine (mercaptan) lyase (O-acetylserine sulfhydrylase, OASS) is one of the important biological enzymes in sulfate assimilation pathway and cysteine synthesis of E. coli, and serine acetyltransferase (Serineacetyltransferase, SAT) exists in the form of enzyme complex. Commonly known as cysteinase (Cysteinesynthase). In microorganisms and plant cells, OASS and SAT reduced inorganic sulfur in the environment to-2 valence sulfur, and synthesized L-cysteine from L-serine by two-step enzyme catalysis. O-acetylserine (OAS) and coenzyme A were synthesized with serine and acetylcoenzyme A as substrate under the catalysis of SAT, and then OAS was converted to cysteine by sulfides catalyzed by OASS. By replacing the substrate in the second step, OASS can synthesize non-protein amino acids from various nucleophilic reagents. Non-protein amino acids are 20 kinds of amino acids other than 20 kinds of amino acids found in proteins existing in nature. Because they enrich the diversity of peptide chains, they provide the basic materials for artificial synthesis of proteins across 20 kinds of natural amino acid barriers. It is the main component of synthetic peptides and peptides at home and abroad, and it is also an attractive candidate for chiral drugs. It is a key intermediate of many new drugs and is widely used in scientific research and medicine. In this paper, the cysK ORF gene fragment of O-acetylserine (mercaptol) lyase A (OASS-A) gene was amplified by PCR using E. coli Escherichia coli (E. coli) DH5 伪 genomic DNA as template, and the fragment of Oacetylserine (mercaptol) lyase A (OASS-A) gene was amplified by PCR. The target fragment was connected with the expression vector pET-22b (), and the recombinant plasmid CysK-pET-22b (), was transformed into the expressing strain. The induced expression conditions were screened and the protein was purified to obtain the high concentration soluble OASS-A recombinant protein. Using O-acetylserine (OAS) as substrate, the enzyme activity of the recombinant protein was detected. The activity unit of the purified recombinant protein was 750 U 路mg, and the active unit of the recombinant protein in the crude extract of the enzyme was 400 U 路mg 路L ~ (- 1). Using recombinant OASS-A as enzyme catalyst and O-acetylserine and phenylthiophenol as substrate, non-protein amino acid S-phenyl-L-cystine (S-P-C) was synthesized. The structure of the product was identified by high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). An effective model for the efficient synthesis of non-protein amino acids by obtaining a large number of active and stable recombinant enzymes as biocatalysts was established. According to the principle of drug splicing, S-P-C was coupled with the effective groups of active compounds asiatidic acid and deoxycholic acid, the structure of the product was identified, and the effect of the synthesized product on the proliferation of cancer cells was preliminarily detected. The results of MTT test showed that the synthesized end products had certain inhibitory effect on proliferation.
【学位授予单位】：辽宁师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914
本文编号：2476805
[Abstract]:O-acetylserine (mercaptan) lyase (O-acetylserine sulfhydrylase, OASS) is one of the important biological enzymes in sulfate assimilation pathway and cysteine synthesis of E. coli, and serine acetyltransferase (Serineacetyltransferase, SAT) exists in the form of enzyme complex. Commonly known as cysteinase (Cysteinesynthase). In microorganisms and plant cells, OASS and SAT reduced inorganic sulfur in the environment to-2 valence sulfur, and synthesized L-cysteine from L-serine by two-step enzyme catalysis. O-acetylserine (OAS) and coenzyme A were synthesized with serine and acetylcoenzyme A as substrate under the catalysis of SAT, and then OAS was converted to cysteine by sulfides catalyzed by OASS. By replacing the substrate in the second step, OASS can synthesize non-protein amino acids from various nucleophilic reagents. Non-protein amino acids are 20 kinds of amino acids other than 20 kinds of amino acids found in proteins existing in nature. Because they enrich the diversity of peptide chains, they provide the basic materials for artificial synthesis of proteins across 20 kinds of natural amino acid barriers. It is the main component of synthetic peptides and peptides at home and abroad, and it is also an attractive candidate for chiral drugs. It is a key intermediate of many new drugs and is widely used in scientific research and medicine. In this paper, the cysK ORF gene fragment of O-acetylserine (mercaptol) lyase A (OASS-A) gene was amplified by PCR using E. coli Escherichia coli (E. coli) DH5 伪 genomic DNA as template, and the fragment of Oacetylserine (mercaptol) lyase A (OASS-A) gene was amplified by PCR. The target fragment was connected with the expression vector pET-22b (), and the recombinant plasmid CysK-pET-22b (), was transformed into the expressing strain. The induced expression conditions were screened and the protein was purified to obtain the high concentration soluble OASS-A recombinant protein. Using O-acetylserine (OAS) as substrate, the enzyme activity of the recombinant protein was detected. The activity unit of the purified recombinant protein was 750 U 路mg, and the active unit of the recombinant protein in the crude extract of the enzyme was 400 U 路mg 路L ~ (- 1). Using recombinant OASS-A as enzyme catalyst and O-acetylserine and phenylthiophenol as substrate, non-protein amino acid S-phenyl-L-cystine (S-P-C) was synthesized. The structure of the product was identified by high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). An effective model for the efficient synthesis of non-protein amino acids by obtaining a large number of active and stable recombinant enzymes as biocatalysts was established. According to the principle of drug splicing, S-P-C was coupled with the effective groups of active compounds asiatidic acid and deoxycholic acid, the structure of the product was identified, and the effect of the synthesized product on the proliferation of cancer cells was preliminarily detected. The results of MTT test showed that the synthesized end products had certain inhibitory effect on proliferation.
【学位授予单位】：辽宁师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914
【参考文献】
相关期刊论文 前2条
1 赵龙铉;田明珠;金礼吉;贺兴隆;沈平;张晓翠;杨君微;;积雪草酸衍生物的合成与表征及体外抗癌活性的研究[J];有机化学;2011年05期
2 Maria J Perez;Oscar Briz;;Bile-acid-induced cell injury and protection[J];World Journal of Gastroenterology;2009年14期
本文编号：2476805
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